Covalent immobilisation of antibodies in Teflon-FEP microfluidic devices for the sensitive quantification of clinically relevant protein biomarkers

Abstract

This study reports for the first time the sensitive colorimetric and fluorescence detection of clinically relevant protein biomarkers by sandwich immunoassays using the covalent immobilisation of antibodies onto the fluoropolymer surface inside Teflon®-FEP microfluidic devices. Teflon®-FEP has outstanding optical transparency ideal for high-sensitivity colorimetric and fluorescence bioassays, however this thermoplastic is regarded as chemically inert and very hydrophobic. Covalent immobilisation can offer benefits over passive adsorption to plastic surfaces by allowing better control over antibody density, orientation and analyte binding capacity, and so we tested a range of different and novel covalent immobilisation strategies. We first functionalised the inner surface of a 10-bore, 200 μm internal diameter FEP microcapillary film with high-molecular weight polyvinyl alcohol (PVOH) without changing the outstanding optical transparency of the device delivered by the matched refractive index of FEP and water. Glutaraldehyde immobilisation was compared with the use of photoactivated linkers and NHS-ester crosslinkers for covalently immobilising capture antibodies onto PVOH. Three clinically relevant sandwich ELISAs were tested against the cytokine IL-1β, the myocardial infarct marker cardiac troponin I (cTnI), and the chronic heart failure marker brain natriuretic peptide (BNP). Overall, glutaraldehyde immobilisation was effective for BNP assays, but yielded unacceptable background for IL-1β and cTnI assays caused by direct binding of the biotinylated detection antibody to the modified PVOH surface. We found NHS-ester groups reacted with APTES-treated PVOH coated fluoropolymers. This facilitated a novel method for capture antibody immobilisation onto fluoropolymer devices using a bifunctional NHS-maleimide crosslinker. The density of covalently immobilised capture antibodies achieved using PVOH/APTES/NHS/maleimide approached levels seen with passive adsorption, and sensitive and quantitative assay performance was achieved using this method. Overall, the PVOH coating provided an excellent surface for controlled covalent antibody immobilisation onto Teflon®-FEP for performing high-sensitivity immunoassays.

Capillary-Driven Flow Microfluidics Combined with Smartphone Detection: An Emerging Tool for Point-of-Care Diagnostics

“…In contrast, fluoropolymer microcapillaries (FEP) have been coated with reagents to achieve hydrophilic surface inside the capillaries which can load aqueous samples rapidly ( Figure 2 h) [ 16 , 42 , 43 , 44 , 45 ]. Pivetal et al [ 42 ] converted the surface of FEP microcapillaries by coating with a layer of polyvinyl alcohol (PVA) and cross-linked reagents for analyte detections (colorimetric or fluorescent), such as the detection of prostate-specific antigen (PSA) [ 43 , 44 ] and cytokines [ 45 ]. These FEP capillaries were injected with multiple solutions, such as PSA standard, detection antibodies, enzyme complex, washing solutions and enzymatic substrates, which were injected in all channels simultaneously.…”

High Dynamic Range Bacterial Cell Detection in a Novel “Lab-In-A-Comb” for High-Throughput Antibiotic Susceptibility Testing of Clinical Urine Samples

“…This material comprises a plastic ribbon containing an array of 10 capillaries along its length with an average diameter of 206 ± 12.6µm and external dimensions of 4.5 ± 0.10 mm wide by 0.6 ± 0.05 mm thick. This material was coated to make internally hydrophilic for production of dipstick microfluidic devices by incubation with a 5 mg/mL solution of PVOH in water (MW 146,000-186,000, >99% hydrolysed, Sigma-Aldrich, UK) at room temperature for a minimum of 2h (26). The material was then washed manually to remove crosslinker with 5 ml of PBS with 0.5 % Tween 20 (Sigma-Aldrich, UK) solution to remove uncoated PVOH, and dried with multiple changes of air using a 50 mL syringe.…”

“…Firstly, even faster detection of growth is needed, with colorimetric resazurin detection known to be hard to colorimetrically quantify by digital imaging due to complex changes in visible light absorbance (27). Faster detection may be achieved by switching to fluorimetric growth detection, previously used by our group for immunoassays in MCF (26). Secondly, a better understanding of the predictive value of direct urine AST testing (without overnight plating and colony isolation) in clinical samples is vital.…”

Design and Fabrication of Capillary-Driven Flow Device for Point-Of-Care Diagnostics

“…Glass/hydrophilic capillaries have also been used to drive flow via capillary action, as described by Lapierre et al [17], who used bare glass capillaries to collect blood samples. In contrast, fluoropolymer microcapillaries (FEP) have been coated with reagents to render their surface hydrophilic and draw up blood or aqueous samples in a minute fraction of time [18][19][20][21][22]. Pivetal et al [19] coated FEP capillaries with polyvinyl alcohol (PVA) to convert their surface from hydrophobic to hydrophilic and attached reagents or antibodies on the surface for the detection of protein biomarkers.…”

“…In contrast, fluoropolymer microcapillaries (FEP) have been coated with reagents to render their surface hydrophilic and draw up blood or aqueous samples in a minute fraction of time [18][19][20][21][22]. Pivetal et al [19] coated FEP capillaries with polyvinyl alcohol (PVA) to convert their surface from hydrophobic to hydrophilic and attached reagents or antibodies on the surface for the detection of protein biomarkers. The reagents reacted with the biomarkers, generating a colour or fluorescent signals which were detected under a microscope attached to a camera.…”